Human growth hormone (hereinafter referred to as ‘hGH’) is a polypeptide hormone consisting of 191 amino acids having a molecular weight of about 22,000 Da being secreted from the anterior pituitary gland. A human growth hormone has been used for the treatment of pediatric pituitary dwarfism mostly. Previously, hGH extracted from human pituitary gland was used, but its supply is limited. Thus only very limited number of people could be treated. Also, after the incidence of degenerative neurological disorder Creutzfeldt-Jacob disease was reported for some of the patients who received the hGH collected from pituitary gland, a use of hGH extracted from pituitary glands has been banned. Currently with the development of genetic engineering techniques, production of hGH in E. coli and yeast has become possible. Thus the biosynthetic hGH medicines produced by genetic engineering have become commercially available through passing toxicological and clinical tests and being approved in several countries after 1985.
In general, polypeptides like hGH have a low stability and thus easily denatures. Also they are readily degraded by serum proteases and removed by kidney or liver. Thus, protein drugs containing polypeptides as pharmaceutical ingredient have to be frequently administered to patients in order to maintain its blood concentration and titer. However, since the protein drugs are often administered in a form of injection, frequent injection of protein drugs to maintain the optimal blood concentration of active polypeptides causes a lot of pain to patients. To solve these problems, there have been many attempts to increase the stability of protein drug in blood and maintain its blood concentration at high level for a long period of time in order to maximize the therapeutic effects of medicine. For the development of a long-acting formulation of the protein drug, one has to make sure that the formulation increases the stability of protein drug while maintaining sufficiently high titer of the drug itself, without causing any immune response in patients.
As a method to stabilize proteins and prevent them from contacting protease and renal loss, a highly soluble polymer such as polyethylene glycol (PEG) has been chemically added to surface of protein drugs. PEG binds to a specific site of a target protein or various sites non-specifically and increases the protein solubility, thereby stabilizing the protein. Also, PEG is effective in prevention of hydrolysis of protein and does not cause any particular side-effects. Although PEG binding may increase the protein stability, a titer of physiologically active protein becomes significantly low, and as the molecular weight of PEG increases, its reactivity with protein is reduced, thereby reducing a yield.
As an alternative method for increasing in vivo stability of physiologically active proteins, a method of producing fusion protein has been developed by connecting a gene encoding for protein with high stability in blood and a gene for physiologically active protein through genetic recombination and culturing the animal cells that are transformed with the recombinant genes. For instance, albumin or its fragment that are known to be highly effective in increasing protein stability can be linked to a targeted physiologically active protein through gene recombination for producing a fusion protein (International Patent Publication Nos. WO 93/15199 and WO 93/15200, European Patent Publication No. EP 413,622).
In addition, as a method for using immunoglobulin, U.S. Pat. No. 5,045,312 discloses that when hGH is conjugated with bovine serum albumin (BSA) or murine immunoglobulin by using a cross-linking agent, activity of the conjugated hGH is increased compared to the unmodified growth hormone. However, the above patent only discloses low-molecular weight compounds such as carbodiimide or glutaraldehyde as a cross-linking agent. When these low-molecular weight cross-linking agents are used, it is hard to obtain homogeneous compositions due to non-specific conjugation, and it may be toxic in vivo. Further, the above-referenced patent merely discloses the increase of growth hormone activity by chemical coupling, but did not describe the effect of chemical coupling on the activity of other types of polypeptide drugs, with no understanding of the correlation with the stability of proteins such as the increase in durability and serum half-life.
Recently, Korean Patent No. 10-0567902 (Physiologically Active Polypeptide Conjugate Having Improved In Vivo Durability) and Korean Patent No. 10-0725315 (Protein Complex Using An Immunoglobulin Fragment And Method For The Preparation Thereof) disclose conjugates prepared by linking physiologically active polypeptides with an immunoglobulin Fc region and a non-peptide polymer, as long-acting formulations of protein drugs which allow for both a minimal reduction of protein activity and an increase in protein stability. According to these methods, hGH may be used as a physiologically active polypeptide to prepare a long-acting hGH conjugate. For commercializing the drug containing long-acting hGH conjugates, it is essential to prevent physicochemical changes such as denaturation, aggregation, adsorption, or hydrolysis due to degradation induced by light, heat or impurities in additives during a storage and transport process, while retaining in vivo effect of hGH. Compared to a hGH polypeptide, a long-acting hGH conjugate has a larger size and increased molecular weight, and thus there is a difficulty in stabilizing the conjugate.
In a pharmaceutical industry, low protein stability in liquid has been a limitation. Thus proteins have been freeze-dried to solve the stability issue. Although a freeze-dried protein formulation has an advantage of retaining stability for a long period of time, when injected it has to be reformulated with dissolution aids. Also, when it is reformulated with dissolution aid or freeze-dried, there were disadvantages in that its activity is often lost due to formation of polymers etc, and the cost and time required for freeze-drying process was high as well. In order to overcome these limitations, International Patent Publication No. WO93/019776 and No. WO94/003198 disclose a stable liquid formulation of hGH. For development of a stable liquid hGH formulation, it is important to control the degradation of hGH such as regulation of production rate of degradation products from deamidation, polymer formation, and oxidation process. Temperature, pH, and additives of liquid formulation affect the production rate of the degradation product, but the formulation that can stabilize all types of proteins allowing for its clinical application has not been known yet. Furthermore, the liquid formulation that has a stabilizing effect for specific protein is often not applicable for stabilization of other proteins. In this regard, the above references disclose that it is first required to perform the selection and combination of factors and additives that can minimize the production rate of specific degradation product of protein in a broad manner to maximize the stability of hGH protein in liquid.
Furthermore, different proteins may be gradually inactivated under different ratio and conditions during storage due to difference in their chemical properties. That is, effects of a stabilizer for extending the storage period are not the same for different proteins. For this reason, the stabilizers used for storage stability have various suitable ratio, concentration, and types according to the physicochemical properties of a target protein. However, when different stabilizers are used simultaneously, it may cause contrary results due to competitive interaction between them and side effects. Furthermore, as properties and concentration of the stored protein may change during its storage, the stabilizers may show different effects from expected. Thus, stabilization of protein in solution takes a lot of efforts and cautions.
Particularly, since long-acting hGH conjugates with increased in vivo durability and stability are prepared by linking a physiologically active peptide hGH with immunoglobulin Fc regions and have significantly different molecular weight and volume from other typical hGH, they require special composition for stabilizing the protein. Also, each of the physiologically active peptide hGH and immunoglobulin Fc region has different physicochemical properties, they both need to be stabilized simultaneously. However, as described above, different peptides or proteins may be gradually inactivated under different ratio and conditions during storage due to the difference in their physicochemical properties. Also, when stabilizers suitable for each peptide or protein are used simultaneously, it may cause contrary results due to competitive interaction between them and side effects. Furthermore, as properties and concentration of the stored protein may change during its storage, the stabilizers may show different effects from expected. Therefore, for a long-acting hGH conjugate, it is difficult to find a stabilizer formulation that can stabilize both of a physiologically active peptide hGH and immunoglobulin Fc region simultaneously.